Folliculogenesis

ABSTRACT

The present invention relates to the use of GDF-9 in assisted reproduction and to kits comprising GDF-9. A method is provided to stimulate follicle and oocyte development and maturation. The method comprises administering GDF-9 in combination with gonadotropins.

The present invention relates to the use of Growth DifferentiationFactor-9 (GDF-9) in assisted reproduction and to kits comprising GDF-9.

Reproduction involves the growth and selection of ovarian follicles,leading to the ovulation of one healthy (high quality) oocyte that canbe fertilised. Subsequently the fertilised oocyte develops to an embryo,which implants in a receptive endometrium and grows to a foetus andfinally a healthy baby. Couples with fertility problems, however, needto undergo treatment in order to obtain children. At the moment twomajor treatment programs are available: Ovulation Induction (OI), forcouples with female infertility due to anovulatory cycles, and In VitroFertilisation (IVF) for couples with either female or male fertilityproblems. In the case of OI protocols, low doses of gonadotropins areadministered in order to induce mono-follicular growth and the ovulationof one oocyte that can be fertilised naturally. In the case of IVFrelatively high levels of gonadotropins are administered in order toobtain multi-follicular development. After the induction ofmulti-follicular growth, oocyte maturation is induced with LuteinizingHormone (LH)/human Chorionic Gonadotropin (hCG) and mature oocytes areretrieved by follicular puncture. After in vitro fertilisation and invitro early embryo cleavage, 2 to 3 embryos are transferred into theuterus.

In both treatments, the gonadotropin dosing has to be adapted toindividual patients in order to obtain mono- or multi-folliculardevelopment. This is extremely difficult, and success is not guaranteed.Moreover, final pregnancy outcome rates are relatively low (˜20%) due tomany factors, among which oocyte quality and endometrium receptivity.The cause of the relatively low success rates may lay in the wayfolliculogenesis and oogenesis are induced.

During the natural menstrual cycle, the gonadotropins FSH and LH inducefollicle and oocyte development. Due to selection, which most likely isalso controlled by FSH, only one follicle from the growing follicle poolbecomes dominant and finally releases a healthy, fertilisable oocyte atovulation. The other follicles become atretic and never will ovulate butdegenerate.

It is well known that not only gonadotropins are needed for optimalfollicle and oocyte development, but also growth factors. GDF-9 is sucha growth factor. Its nucleotide and amino acid sequence has beendescribed by Incerti et al (Biochim.Biophys.Acta (1994), 1222, 125-128)and McGrath et al (Mol. Endo. (1995), 9, 131-136). In GDF-9 knockoutmice it has been demonstrated that follicular development ceases at theprimary stage due to GDF-9 deficiency (Dong et al., 1996). Moreover ithas been demonstrated that GDF-9 is present in follicles (in the oocytesto be more specific) from the primary stage up to the fully grownpreovulatory stage follicles (McGrath et al., 1995). These resultsindicate that GDF-9 is involved in normal follicle and oocytedevelopment.

Due to selection mechanisms only one follicle from the group offollicles that left the primordial pool, reaches the preovulatory stage,i.e. the dominant follicle, and provides a healthy, fertilizable oocyte.The others become atretic and degenerate. The mechanisms controlling theselection of a dominant follicle are not fully understood, but is thehypothesis is that the follicle that is most sensitive to FSH, is theone that becomes dominant.

Follicular growth thus is controlled by growth factors such as forexample IGF-1, GDF-9, and later on by the gonadotropins FSH and LH, andby estrogens. During growth several stages can be identified: theprimary stage, the preantral and antral stage, and finally thepreovulatory stage.

A major difference between infertility treatment protocols and naturalconception is the number of follicles and oocytes that are involved.

During Assisted Reproduction Techniques (ART) treatment, the selectionmechanism that leads to the ovulation of one healthy oocyte that will befertilised and will develop to an implanting embryo, is overruled byadministration of relatively high gonadotropin (mainly FSH) doses. Thisprotocol causes many follicles and oocytes to grow and finally manyoocytes are obtained from these follicles. However, although oocytematuration is induced with LH/CG, not all oocytes actually have maturedat the moment of retrieval, or oocytes have matured but do not developproperly. In some cases, oocyte maturation can subsequently be inducedin vitro, but this in vitro maturation procedure has limited success. Asa result only a relatively small proportion of all oocytes obtained fromthe follicles will eventually develop to an embryo that implants in theendometrium. This leads to relatively low ART success.

The present invention provides for an improvement of stimulatingfollicle and oocyte development and maturation by administration ofGDF-9 in combination with gonadotropins. This will lead to the retrievalof higher quality oocytes with higher fertilisation, cleavage andimplantation capacities and thus finally to higher ART success rates.Thus, according to the present invention GDF-9 might be used in therapy.Especially, it might be used for assisted reproduction.

Thus, due to administration of GDF-9, smaller follicles, which are notdestined to become apoptotic, will be selected into the growing pool,and will grow to healthy preovulatory follicles. in addition, growingfollicles might be rescued by GDF-9 and will develop to a fully grownfollicle with a high quality oocyte, leading to an increase in finalsuccess rates, i.e. pregnancy outcome.

The induction of follicular growth by a combination of the growth factorGDF-9 and gonadotropins might lead to a more natural folliculardevelopment, and herewith might improve oocyte quality and thus finalsuccess rate.

According to the present invention GDF-9 can be administered to femalepatients in order to improve follicle and oocyte development andmaturation. The administration can be included in existing treatmentprotocols for ovarian stimulation and in vitro fertilisation.Alternatively, GDF-9 can also be used for developing isolated folliclesand/or oocytes by incubation in vitro in a suitable culture medium inthe presence of GDF-9, preferably in addition to FSH.

GDF-9 preferably is prepared by recombinant DNA technology in eukaryotichost cells. DNA encoding GDF-9 has been isolated and characterized andcan be used for the preparation of suitable vector system. GDF-9preferably is encoded by DNA of mammalian origin, more preferably byhuman DNA.

The nucleotide acid sequence of GDF-9 of one species can be used as aprobe or as a source to prepare synthetic oligonucleotides to be used asprimers in DNA amplification reactions allotting the isolation andidentification of complete gene of other species. Using that sequenceinformation, the complete gene can be derived from cDNA or genomic DNAfrom other sources or synthesized using known methods.

Under gonadotropins is to be understood FSH as isolated from humansources, such as menopausal urine. Alternatively, recombinant FSH mightbe prepared by production in eukaryotic host cell. Furthermore, as incurrent assisted reproduction protocol treatments, maturation can beinduced by the administration of LH or hCG. However, also variationsthereupon including GnRH antagonists and/or agonists can be used.

The proteins to be used according to the invention are those proteinswhich have the amino acid sequences as isolated from the relevantvertebrate tissue, and have these known sequences per se, or theirallelic variants thereof.

The variations that can occur in a sequence may be demonstrated by (an)amino acid difference(s) in the overall sequence or by deletions,substitutions, insertions, inversions or additions of (an) amino acid(s)in said sequence. Amino acid substitutions that are expected not toessentially alter biological and immunological activities, have beendescribed. Amino acid replacements between related amino acids orreplacements which have occurred frequently in evolution are, inter aliaSer/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Davhof, M. D., Atlas ofprotein sequence and structure, Nat. Biomed. Res. Found., WashingtonD.C., 1978, vol. 5, suppl. 3).

It is to be understood that according to the present invention alsofragments of GDF-9 or splice variants can be used as long as theessential part of the protein is still intact, that is as long as thevariant protein still exerts its function in follicle and oocytedevelopment. Follicle and oocyte development can be mimicked in vitrousing a mouse in vitro follicle culture system. In that systempre-antral follicles can be grown to the preovulatory stage.

The gonadotropins to be used in combination with GDF-9 according to theinvention can be dimeric i.e. composed of two non-covalently boundsubunits. They can, however, comprise modifications generally known inthe art.

In one such modification of the gonadotropins, the C-terminus of theamino acid sequence of one of the subunits is linked, optionally througha linker moiety, to the N-terminus of the amino acid sequence of theother subunit. Preferably the linker moiety is a complete or partial CTPunit or variant thereof, or a repeated oligopeptide e.g. a 5 timesrepeated Ser-Gly peptide.

Another modification can be an extension of the α and/or β subunit attheir respective N- or C-termninus with a complete or partial CTP unitor a variant thereof. The extension may comprise the respective CTPunits in single or multiple forms. Alternatively, a complete CTP unit orpartial CTP unit or multiple forms thereof can be inserted in the N- orC-termninus of said subunits. Again another modification is theintroduction of one or more non-native disulfide bridges. Suchextensions may also be added to GDF-9 in order to modify the half-lifeof the protein.

Furthermore, the proteins may be either glycosylated or partiallyglycosylated. Partially glycosylated proteins to be used according tothe invention can be obtained by site-directed mutagenesis whereby oneor more of the glycosylation recognition sites are removed.Alternatively, the glycosylation pattern can be modified by theintroduction of additional glycosylation recognition sites and,optionally, the removal of one or more glycosylation recognition sites,resulting in a modified glycosylation of said proteins. A glycosylationrecognition site as used herein consists of the amino acid sequenceAsn-X-Ser/Thr, wherein X can be any amino acid.

As used herein, the “CTP unit” refers to the amino acid sequence foundat the carboxy terminus of the β subunit of hCG which extends from aminoacid 112-118 to residue 145 at the C-terminus or to a portion thereof. A“complete” CTP unit contains 28-34 amino acids, depending on theN-terminus of the CTP. A “partial” CTP unit is an amino acid sequencewhich occurs between positions 112-118 to 145 inclusive, but which hasat least one amino acid deleted from the shortest possible complete CTPunit (amino acid 118-145). “Multiple” CTP units are understood toencompass tandem arrays of the complete CTP unit or partial CTP unit orcombinations of both.

Methods to construct GDF-9 and the gonadotropins are well known in theart (Sambrook et al., Molecular Cloning: a Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, latest edition). Themost practical approach is to produce these proteins by expression ofthe DNA encoding the desired protein. Techniques for site directedmutagenesis, ligation of additional sequences, PCR, and construction ofsuitable expression systems are all, by now, well known in the art.Portions or all of the DNA encoding the desired proteins can beconstructed synthetically using standard solid phase techniques,preferably to include restriction sites for case of ligation. Suitablecontrol elements for transcription and translation of the includedcoding sequence can be provided to the DNA coding sequences. As is wellknown. expression systems are now available which are compatible with awide variety of hosts. including prokaryotic hosts such as bacteria andeucaryotic hosts such as yeast, plant cells, insect cells, mammaliancells, avian cells and the like. The choice of host is particularly topost-translational events, most particularly including glycosylation.The location of glycosylation is mostly controlled by the nature of theglycosylation site within the molecule. However, the nature of thesugars occupying this site is largely controlled by the nature of thehost. Most preferred hosts are from mammalian origin. Most preferablythe cell is a CHO cell line. Chinese Hamster Ovary (CHO) cells have beentransfected with human gonadotropin subunit genes and these cells areshown to be capable of secreting intact dimers (e.g. Keene et al (1989),J.Biol.Chem., 264, 4769-4775; Van Wezenbeek et al (1990), in From cloneto Clinic (eds Crommelin D. J. A. and Schellekens H.), 245-251).

Various stimulation protocols can be applied. There are great inter- andintra-individual variations in the response of the ovaries to exogenousproteins. This makes it impossible to set a uniform dosage scheme. Thedosage should, therefore, be adjusted individually depending on theovarian response. This requires ultrasonography and monitoring ofoestradiol levels. Protocols to be used for anovulation and controlledovarian hyperstimulation are generally known. Depending on the treatmentFSH administration is started at day 1, 2 or 3 and may be continued forup to 5-12 days. GDF-9 administration can be started simultaneous withFSH or 1-3 days prior to the FSH administration. The GDF-9administration can then be stopped or continued in combination with FSH.Ovulation and/or oocyte maturation can be induced by the administrationof hCG or LH. To prevent premature luteinization in addition to FSH alsoGnRH agonists can be administered. In patient undergoing COH prematureLH surges may also be prevented by GnRH antagonist administration.

In cases where GDF-9 and FSH are jointly administered GDF-9 can be mixedwith FSH, however, it might also be administered separately atapproximately the same time.

Ovarian response is monitored by ultrasonography and measurement ofplasma oestradiol levels. This is performed for ovulation induction aswell as for COH. Typically, in case of COH, when ultrasonographicevaluation indicates the presence of at least three follicles of 16-20mm, and there is evidence of a good oestradiol response (plasma levelsof about 300-400 pg/ml (1000-1300 pmol/l) for each follicle with adiameter greater than 18 mm), the final phase of maturation of thefollicles can be induced by administration of hCG. Oocyte retrieval isperformed 34-35 hours later.

It is to be understood that variations of the general stimulation schemeincluding the uses of FSH, hCG, LH GnRH antagonists and GnRH agonists orthe like are all part of this invention as long as the administration ofGDF-9 is integrated in such a protocol.

Thus, according to the present invention it has been found that currentinfertility treatment protocols can be optimized by combined in vivoadministration of GDF-9 and gonadotropins. Furthermore, initiation offollicular growth with GDF-9, followed by gonadotropin treatment,optimises follicular development and herewith oocyte quality.

The initiation of follicular growth with GDF-9 might also be followed bycombined administration of GDF-9 and gonadotropins.

According to an other aspect of the invention follicles and oocytes canbe developed in vitro by incubation in a chemically defined culturemedium comprising GDF-9 and gonadotropins.

According to an other aspect of the invention there is provided a kitfor stimulating follicle and oocyte development and maturation. Such akit may comprise several package units. Such units may comprise GDF-9admixed with gonadotropins. Alternatively GDF-9 and the gonadotropinsmight also be packaged seperately. Under gonadotropins is to beunderstood FSH. Optionally also separate package units might be presentcontaining hCG or LH.

The amount of GDF-9 per ampoule might change from 10 ng-500 μg,preferably from 200 ng-200 μg. The range of FSH usually is between50-500 IU.

For in vitro development a suitable amount of GDF-9 is an amount of2-500 ng/ml, whereas FSH is used in range from 50 mIU-1 IU.

The pharmaceutical preparations for use according to the invention canbe prepared in accordance with standard techniques such as for exampleare described in the standard reference, Gennaro et al. (Ed.),Remmington's Pharmaceutical Sciences, (18 th ed. Mack PublishingCompany, 1990, e.g. Part 8: Pharmaceutical Preparations And TheirManufacture). For the purpose of making the pharmaceutical preparationsaccording to the invention, the active substance is mixed with ordissolved in a pharmaceutical acceptable carrier.

Any conventional pharmaceutical carrier that does not interfere withperformance of the active ingredient can be used in the preparationsaccording to the present invention.

Pharmaceutical acceptable carriers are well known to those skilled inthe art and include, for example, sterile salin, lactose, sucrose,calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, oliveoil, sesame oil and water.

Furthermore the pharmaceutical composition according to the inventionmay comprise one or more stabilizers such as, for example, carbohydratesincluding sorbitol, mannitol, starch, sucrosedextrin and glucose,proteins such as albumin or casein, and buffers like alkalinephosphates.

Suitable administration routes are intramuscular injections,subcutaneous injections, intravenous injections or intraperitonealinjections, oral and intranasal administration.

LEGENDS TO THE FIGURES

FIG. 1

The effect of gonadotropins and GDF-9 on mouse follicular growth.

EXAMPLES Example 1 Test of GDF-9 in an in vitro Follicle Culture

During follicular growth, various developmental stages are passedranging from the primordial, primary, pre-antral, antral and finally thepreovulatory stage. The proliferation and differentiation processesneeded for optimal follicular development, are controlled bygonadotropins, steroids and growth factors. During infertilitytreatment, follicles of the early-antral stage are induced to grow withgonadotropins. This process can be mimicked in vitro using a mouse invitro follicle culture system. In addition it is possible to startfolliculogenesis in vitro at an earlier stage: the preantral orsecondary stage.

Materials and Methods of the in vitro Follicle Culture

Follicle Culture

Female immature mice (F1: B6 BCA; 21-23 days of age) are anaesthetisedwith ether and blood is collected by means of eye extraction. Afterclotting, blood is centrifuged for 15 min. at 4000 g and serum iscollected and stored at −20° C. until use.

Ovaries are removed and placed in Leibovitz-L15 medium (Gibco, Paisley,UK; #11415-049) supplemented with glutamin (2 mM: Gibco, #15039-019),transferrin (10 μg/ml: Sigma, St. Louis, Mo., USA; T-5391), insulin (5μg/ml: Sigma, I-1882), ascorbic acid (50 μg/ml: Sigma, A-4034), selenium(2 ng/ml: Sigma, S-9133) and Bovine Serum Albumin (0.3%, BSA: Sigma,A-9647), at 37° C. Preantral follicles with a diameter of 150 to 180 μmare isolated with two 30 Gauge×½ needles attached to 1 ml syringes andcollected in αMEM medium (Gibco, #22571-020) supplemented with glutamin(2 mM), transferrin (10 μg/ml), insulin (5 μg/ml), ascorbic acid (50μg/ml), selenium (2 ng/ml) (i.e. αMEM culture medium) and BSA (0.3%).Isolated follicles are incubated in is a humidified incubator gassedwith 5% CO₂ in air at 37° C. until enough follicles are isolated for theculture. From the collected follicles, follicles with normalmorphological appearance i.e. a central spherical oocyte, high densityof granulosa cells and a theca cell layer enclosing the entire follicle,are selected and individually cultured in Millicell-CM culture plateinserts (Millipore, Bedford, USA; #PICM 01250) with 250 μl αMEM culturemedium supplemented with 5% immature mouse serum with or without GDF-9containing human embryonic kidney 293 T cell culture supernatant. As acontrol, follicles are cultured with 293 T cell culture supernatant notexpressing GDF-9. Follicles are subsequently cultured in a humidifiedincubator gassed with 5% CO, in air at 37° C. After 20 h of culture, 200μl medium is exchanged with fresh medium in addition with 100 mU/mlrecombinant human Follicle Stimulating Hormone (recFSH: NV Organon, Oss,The Netherlands) to induce follicular growth, or still without recFSH.Culture medium is exchanged every other day and the diameter of thefollicles is measured each day using 100× magnification and a calibratedmicrometer.

Follicular Growth

Mouse follicles of approximately 170 μm are cultured in medium with 5%immature mouse serum (IMS) and supernantant of recombinant rat GDF-9transfected human embryonic kidney 293 T cells (200 ng/ml rGDF-9). Afterthe first 24 h of culture, medium was refreshed and follicles werecultured further in the presence (+100 mIU/ml FSH) or absence (−FSH) ofrecombinant human FSH (recFSH). As a control, follicles were culturedwith 20% (v/v) supernantant of non-transfected 293 T cells, also in thepresence or absence of recFSH.

Under the influence of immature mouse serum and recFSH, mouse folliclesgrow to 350 μm within 5 days. The addition of GDF-9 to the recFSHcultures, increases follicular growth rate and follicles reach 350 μmwithin 3 days. After 5 days of culture, follicles reach a size of 450μm. Also in the absence of recFSH GDF-9 increases follicular growthabove the levels reached in the absence of GDF-9.

What is claimed is:
 1. A follicular growth stimulating composition,comprising: an effective amount of Growth Differential Factor-9 (GDF-9)to stimulate follicle growth and oocyte development and maturation, aneffective amount of at least one gonadotropin and a pharmaceuticallyacceptable carrier.
 2. A method for stimulating follicle and oocytedevelopment and maturation, comprising: administering to a femalepatient in need thereof an effective amount of Growth DifferentialFactor-9 (GDF-9) and an effective amount of at least one gonadotropin tostimulate follicle and oocyte development and maturation.
 3. A follicleand oocyte development and maturation kit, comprising: a plurality ofpackage units comprising a mixture of at least one gonadotropin andGrowth Differential Factor-9 (GDF-9).
 4. A follicle and oocytedevelopment and maturation kit, comprising: at least one gonadotropinand Growth Differential Factor-9 (GDF-9) in separate package units.
 5. Amethod of assisted reproduction, comprising: administering to a femalepatient an effective amount of Growth Differential Factor-9 (GDF-9) andan effective amount of at least one gonadotropin to stimulate growth offollicles.
 6. The method of claim 2, wherein the GDF-9 is administeredprior to the administration of the at least one gonadotropin.
 7. Themethod of claim 6, wherein the GDF-9 is begun being administered atleast one day prior to the administration of the at least onegonadotropin.
 8. The method of claim 5, wherein the GDF-9 isadministered prior to the administration of the at least onegonadotropin.
 9. The method of claim 8, wherein the GDF-9 isadministered at least one day prior to the administration of the atleast one gonadotropin.